https://github.com/vibtobilab/methylseq
Methylation (Bisulfite-Sequencing) analysis pipeline using Bismark or bwa-meth + MethylDackel
https://github.com/vibtobilab/methylseq
Last synced: 2 months ago
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Methylation (Bisulfite-Sequencing) analysis pipeline using Bismark or bwa-meth + MethylDackel
- Host: GitHub
- URL: https://github.com/vibtobilab/methylseq
- Owner: VIBTOBIlab
- License: mit
- Created: 2025-07-15T15:30:34.000Z (about 1 year ago)
- Default Branch: main
- Last Pushed: 2026-05-19T10:18:55.000Z (2 months ago)
- Last Synced: 2026-05-19T11:35:32.327Z (2 months ago)
- Language: Nextflow
- Homepage:
- Size: 19.9 MB
- Stars: 1
- Watchers: 0
- Forks: 0
- Open Issues: 0
-
Metadata Files:
- Readme: README.md
- Changelog: CHANGELOG.md
- License: LICENSE
- Code of conduct: CODE_OF_CONDUCT.md
- Citation: CITATIONS.md
- Codeowners: .github/CODEOWNERS
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README
# MethylSeq pipeline of TOBI lab
[](https://www.nextflow.io/)
[](https://docs.conda.io/en/latest/)
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[](https://sylabs.io/docs/)
[](https://github.com/askimed/nf-test)
## Introduction
> **Note:** This pipeline has been modified using the original nf-core/methylseq pipeline (version 2.6.0) to include the optical removal duplicates and some other modules in the Bismark subworkflow. You can find the original pipeline at the following [nfcore repository](https://nf-co.re/methylseq/2.6.0).
**MethylSeq** is a bioinformatics analysis pipeline used for Methylation (Bisulfite) sequencing data. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality-control on the results.
The pipeline is built using [Nextflow](https://www.nextflow.io), a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker / Singularity containers making installation trivial and results highly reproducible.
On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources.The results obtained from the full-sized test can be viewed on the [nf-core website](https://nf-co.re/methylseq/results).
## Pipeline Summary
The pipeline allows you to choose between running either [Bismark](https://github.com/FelixKrueger/Bismark) or [bwa-meth](https://github.com/brentp/bwa-meth) / [MethylDackel](https://github.com/dpryan79/methyldackel).
Choose between workflows by using `--aligner bismark` (default, uses bowtie2 for alignment), `--aligner bismark_hisat` or `--aligner bwameth`.
| Step | Bismark workflow | bwa-meth workflow |
| -------------------------------------------- | --------------------- | --------------------- |
| Generate Reference Genome Index _(optional)_ | Bismark | bwa-meth |
| Merge re-sequenced FastQ files | cat | cat |
| Raw data QC | FastQC | FastQC |
| Adapter sequence trimming | Trim Galore! | Trim Galore! |
| Align Reads | Bismark | bwa-meth |
| Filter Non Conversion | Bismark | - |
| Deduplicate Alignments | Bismark | Picard MarkDuplicates |
| Removal of optical duplicates | Picard MarkDuplicates | - |
| Extract methylation calls | Bismark | MethylDackel |
| Sample report | Bismark | - |
| Summary Report | Bismark | - |
| Alignment QC | Qualimap | Qualimap |
| Sample complexity _(optional)_ | Preseq | Preseq |
| CpGs-level saturation _(optional)_ | methurator | methurator |
| Project Report | MultiQC | MultiQC |
## Usage
> [!NOTE]
> If you are new to Nextflow and nf-core, please refer to [this page](https://nf-co.re/docs/usage/installation) on how to set-up Nextflow. Make sure to [test your setup](https://nf-co.re/docs/usage/introduction#how-to-run-a-pipeline) with `-profile test` before running the workflow on actual data.
First, prepare a samplesheet with your input data that looks as follows:
`samplesheet.csv`:
```csv
sample,fastq_1,fastq_2
SRR389222_sub1,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub1.fastq.gz
SRR389222_sub2,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub2.fastq.gz
SRR389222_sub3,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub3.fastq.gz
Ecoli_10K_methylated,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R1.fastq.gz,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R2.fastq.gz
```
Each row represents a fastq file (single-end) or a pair of fastq files (paired end).
Now, you can run the pipeline using:
```bash
nextflow run main.nf --input samplesheet.csv --outdir --genome GRCh37 -profile
```
> [!WARNING]
> Please provide pipeline parameters via the CLI or Nextflow `-params-file` option. Custom config files including those provided by the `-c` Nextflow option can be used to provide any configuration _**except for parameters**_;
> see [docs](https://nf-co.re/usage/configuration#custom-configuration-files).
For more details and further functionality, please refer to the [usage documentation](https://nf-co.re/methylseq/2.6.0/docs/usage), the [parameter documentation](https://nf-co.re/methylseq/2.6.0/parameters) and the section below for the parameters that have been added to the modifed version of the pipeline.
## Parameters included in the modified version
The original [nf-core/methylseq pipeline](https://nf-co.re/methylseq/2.6.0/) has been modified to include modules that are important when processing RRBS/cfRRBS samples. The pipeline has been modified by @edogiuili and is currently maintained by Edoardo Giuili ([@edogiuili](https://github.com/edogiuili)) and Sofie Van de Velde ([@sofvdvel](https://github.com/sofvdvel)).
### Removal of optical duplicates
#### `--remove_optic_duplicates`
If specified, it removes the optical duplicates. It can be used together with `--sequencer` (see below).
#### `--sequencer`
If the flag `--remove_optic_duplicates` has been specified, `--sequencer` will be by default set to **NovaSeq**. Alternatively, you can specify **HiSeq** or **NextSeq**. This will change the OpticalDupsPixelDistance within PicardMarkDuplicates step (**NovaSeq**: 12000, **HiSeq**: 2500, **NextSeq**: 100).
### Filter non conversion (Bismark) module
#### `--filter_non_conversion`
If specified, it filters out all those reads that have a methylation value >= than a preset threshold in non-CG context where you expect a very low methylation level (<5% usually). For more information please consult the [Bismark usage](https://felixkrueger.github.io/Bismark/bismark/filter_nonconverted_reads/).
#### `--minimum_count`
Minimum number of methylation sites for a read to be filtered out (def. 3).
#### `--percentage_cutoff`
Minimum methylation percentage for a read to be filtered out (def. 90%).
### CpG-level sequencing saturation using methurator
#### `--run_methurator`
If specified (by default: true), it will run [methurator](https://vibtobilab.github.io/methurator/latest/) to compute a CpG-level sequencing saturation analysis.
#### `--t_max`
Maximum extrapolation factor (t) value (def. 10).
#### `--minimum_coverage`
Minimum number of counts to define a new CpG (def. 1). More than 1 value can be specified (e.g. '1,3,5').
## Pipeline output
To see the results of an example test run with a full size dataset refer to the [results](https://nf-co.re/methylseq/results) tab on the nf-core website pipeline page.
For more details about the output files and reports, please refer to the
[output documentation](https://nf-co.re/methylseq/output).
## Credits
These scripts were originally written for use at the [National Genomics Infrastructure](https://portal.scilifelab.se/genomics/) at [SciLifeLab](http://www.scilifelab.se/) in Stockholm, Sweden.
- Main authors of original nf-core/methylseq pipeline:
- Phil Ewels ([@ewels](https://github.com/ewels/))
- Maintainers:
- Felix Krueger ([@FelixKrueger](https://github.com/FelixKrueger))
- Sateesh Peri ([@Sateesh_Peri](https://github.com/sateeshperi))
- Edmund Miller ([@EMiller88](https://github.com/emiller88))
- Contributors:
- Rickard Hammarén ([@Hammarn](https://github.com/Hammarn/))
- Alexander Peltzer ([@apeltzer](https://github.com/apeltzer/))
- Patrick Hüther ([@phue](https://github.com/phue/))
## Citations
An extensive list of references for the tools used by the pipeline can be found in the [`CITATIONS.md`](CITATIONS.md) file.
You can cite the `nf-core` publication as follows:
> **The nf-core framework for community-curated bioinformatics pipelines.**
>
> Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.
>
> _Nat Biotechnol._ 2020 Feb 13. doi: [10.1038/s41587-020-0439-x](https://dx.doi.org/10.1038/s41587-020-0439-x).